Device for manipulation of packets in micro-containers, in particular in microchannels

ABSTRACT

A microfluidic device for performing physical, chemical or biological treatment to at least one packet without contamination.

The present invention relates to a device for manipulation of packets inmicro-containers, in particular in microchannels.

As used herein,

packet

refers to compartmentalized matter and may refer to a fluid packet, anencapsulated packet and/or a solid packet.

A fluid packet refers to one or more packets of liquids or gases. Afluid packet may refer to a droplet or bubble of a liquid or gas. Afluid packet may refer to a droplet of water, a droplet of reagent orsample, a droplet of solvent, a droplet of solution, a particlesuspension or cell suspension, a droplet of an intermediate product, adroplet of a final reaction product or a droplet of any material. Anexample of a fluid packet is a droplet of aqueous solution suspended inoil. In a preferred embodiment, a fluid packet refers to a droplet ofwater or a droplet of solution.

An encapsulated packet refers to a packet enclosed by a layer ofmaterial. The surface of an encapsulated packet may be coated with areagent, a sample, a particle or cell, an intermediate product, a finalreaction product, or any material. An example of an encapsulated packetis a lipid vesicle containing an aqueous solution of reagent suspendedin water.

A packet may contain for instance a vesicle or other microcapsule ofliquid or gas that may contain a reagent, a sample, a particle, a deadcell or alive cell, an intermediate product, a final reaction product,or any material.

A solid packet refers to a solid material, for example biologicalmaterial, that may contain, or be covered with, a reagent, a sample, aparticle or cell, an intermediate product, a final reaction product, orany material. An example of a solid packet is a latex microsphere withreagent bound to its surface suspended in an aqueous solution. A packetmay contain a crystal, a polycrystalline material or a vitreousmaterial.

Packets may be made to vary greatly in size and shape, and may have amaximum dimension between about 100 nm and about 1 cm.

Droplet systems may consist in water-based droplets in oil or afluorinated solvent, or “oily” (water immiscible) droplets in an aqueoussolvent. The fluid involved in the droplet system of the invention maybe any kind of fluid, aqueous, organic, mineral, hydrophilic, orhydrophobic, including water based buffers, biological fluids,hydroorganic solvents, Liquids made of molecules with carbon-carbonbackbone, Si—Si backbone (silicone), heteroatom backbone (such as forexample polyethylene glycol), or ionic liquids. Droplet systems havereceived much attention in microfluidics as a method for producingprecise emulsions, as discrete microreactors for polymerase chainreaction (PCR), for the measurement of fast kinetics, and for thedispersion-free transport and manipulation of sample aliquots.Considerable efforts have thus been developed in the last years tocreate and: or manipulate microdroplets. Some devices use hydrophobicforces, by moving such droplets in microchannel combining somehydrophilic and some hydrophobic portions. For instance, U.S. Pat. No.6,130,098 discloses a method for moving microdroplets, comprising:

-   -   providing a microdroplet transport channel having one or more        hydrophobic regions and in communication with a gas source,    -   introducing liquid into said channel under conditions such as        the liquid stops at one of the hydrophobic regions,    -   separating a microdroplet by increasing the pressure applied by        the gas source so as to let such droplet moves over the        hydrophobic region.

This approach imposes that different droplets be in contact with thesame solid surface, and is thus prone to contamination.

Manipulation of droplets on planar arrays of electrodes byelectrowetting has also become very popular, since it allows one toaddress droplets to diverse locations and along complex and programmablepaths. For instance, U.S. Pat. No. 6,294,063 discloses an apparatus forprogrammably manipulating a plurality of packets, such packetsoptionally being droplets, said apparatus comprising a reaction surfaceconfigured to provide an interaction site for such packets, an inletport, means to generate manipulation forces upon said packets, theforces being capable of programmably moving said packets about saidreaction surface along arbitrarily chosen paths, and a position sensor.

U.S. Pat. No. 6,565,727 also discloses a device for manipulating adroplet of a polar liquid, comprising an upper and lower surface,defining between them a gap, said upper surface comprising a pluralityof interdigitated electrodes, and said lower surface comprising a commoncounterelectrode. The device further comprises insulating layers betweensaid electrodes and said gap, and a non-polar liquid positioned in thegap. In this device, a droplet can he maintained on top of a firstelectrode on the upper surface, by applying a potential between saidelectrode and the counterelectrode on the lower surface, making theupper surface wetting for the droplet in the vicinity of said firstelectrode. Then, the droplet can be moved to a second electrode on the,upper surface interdigitated with said first electrode, by suppressingthe potential difference between the first electrode and thecounterelectrode, and applying a potential difference between the secondelectrode and the counterelectrode to make said second electrode wettingto the fluid.

Electrowetting can also be used to mix two different droplets, asdescribed e.g. in M. Washizu, IEEE Trans. Ind. Appl., 34, 732-737(1998). Mixing of droplets containing two reagents or a sample and areagent is a key technological step for developing microfluidicintegrated systems or “lab-on-chips”.

The format of a planar array of electrodes required by electrowetting,however, has severe drawbacks. The fabrication of the array ofelectrodes is complex, and becomes extremely expensive and technicallydemanding for surfaces exceeding a few square cm. Therefore,transporting droplets on large distances, e.g. more than 10 cm, isimpractical. Also, for liquid droplets the surface should be kepthorizontal and relatively vibration free, to avoid unwanted motion ofdroplets under the action of gravity or acoustic waves. Dropletmanipulation in planar format may also he limited by dropletevaporation, the latter being a serious hindrance in quantitativebiochemistry applications, since reaction yields are highly sensitive toconcentration, Electrowetting may further introduce surfacecontamination, Another limitation is that electrowetting can only workwith liquids, so that it cannot be used to transport solid objects.

Dielectrophoresis is another way of transporting and mixing droplets orsolid objects such as cells or latex particles. For instance, Schwartzet al, in Lab Chip, 4, 11-17 (2004) discloses a programmable fluidprocessor, in which droplets can be moved and mixed on top of an arrayof electrodes, by energizing sequentially electrodes in the array. Thismethod, however, also requires a complex array of electrodes on a planarsurface, and thus shares many of the drawbacks of electrowetting. Inanother instance, Velev et al., Nature, 426, 515-516 (2003) describes aprocess for moving and mixing droplets which are floating on a layer offluorinated oil, where the oil is in contact with a pattern ofelectrodes. This eliminates the contamination problems inherent inelectrowetting, but still requires fabricating a complex array ofelectrodes.

Transporting and mixing droplets in an elongated microchannel, or in anetwork of connected microchannels is More robust to the above problems.For instance, it avoids evaporation, and allows transport on longdistances by simple hydrodynamic mobilization of a carrier fluidsurrounding the droplets. Droplets can thus be transported incapillaries several meters long, and used as microreactors, as disclosede.g. in Curcio and Roeraade, Anal. Chem., 75, 1-7 (2003), Wheninteraction with the walls is well controlled, all droplets move at thesame velocity, and very stable trains are achieved. However, the systemsof this kind, suffer from contamination between droplets, which has beenattributed to the formation of small satellite drops in the flow. Parket al. Anal. Chem. 2003, 75, 6029-6033, has proposed a system in whichnumerous aqueous sample plugs are separated by plugs of air. However,wall treatments are not sufficient to completely eliminatecontamination, so it is sometimes necessary to include a “wash” dropletbetween samples. In addition, bubbles in the stream may introduceirregularities in the thermal history of the droplets, making thistechnique less attractive for quantitative applications

It was also proposed to manipulate solid particles or cells inmicrochannels by dielectrophoresis. Dielectrophoresis uses a forceexerted on a particle with a dielectric constant different from that ofthe surrounding medium in a gradient of electric field. Different typesof arrangements were used to date for the application ofdielectophoresis in microchannels. In a first one, an array of closelyspaced interdigitated electrodes locally creates lines of high fieldgradient in which particles are attracted. Optionally, these lines canhe shifted in time by alternately energizing different series ofelectrodes, said method being called “travelling wanedielectrophoresis”, For instance, Schnelle et al., Electrophoresis, 21,66-73 (2000) discloses a method for sorting particles, in which they aredeflected by travelling wave dielectrophoresis between a multiplicity ofelectrodes in an interdigitated arrangement, and energized sequentiallywith a four phase alternating electric signal. By using pairs ofelectrodes of different shapes facing each other across themicrochannel, and applying a potential difference between them, it isalso possible to create different kinds of dielectrophoretic traps,cages or deflecting electrodes, as described e.g. in Durr al.,Electrophoresis, 24, 722-731 (2003).

These dielectrophoretic devices present some advantages upon planarsystems. In particular, they are more robust to tilting or vibration.However, they still require complex microfabrication, and are expensiveto fabricate.

Another key hurdle in the development of microchannel droplet systems,especially for microreactor applications, is the mixing of samples orreagents from different sources. For this, one needs to coalesce twodroplets, but Laplace and hydrodynamic forces tend to make thiscoalescence difficult. When arriving simultaneously at a T-intersection,one drop simply follows the second one into the T without coalescing.Coalescence can be forced by contact charging the droplets, but thiscould be a major source of contamination in PCR and other biologicalsystems. Once introduced into a channel, a smaller droplet trailing alarger one may eventually coalesce with it, since the smaller dropletmoves with a higher average velocity, However, this is not a rationalestrategy microfluidics, since film drainage between the droplets is veryslow, leading to coalescence distances between 30-100 tube diameters(Olbricht and Kung, J. Colloid Interface Sci., 120, 229-244 (1987)).Moreover, under certain conditions (relative droplet sizes, viscosities,etc.) no coalescence is achieved, and the coalescence time or positionare not very reproducible.

Consequently, there exists a strong need for a device and methodproviding reproducible, contamination-free droplet manipulation, inparticular coalescence, in microchannels or closed microfluidic systems.

An object of the present invention among others is to provide suchdevice and method.

The present invention relates to, according to one of its aspects, amicrofluidic device for deforming, in particular splitting, at least onepacket, or displacing at least two packets towards each other, inparticular for collapsing, said device comprising:

-   -   a microchannel having an axis,    -   packet manipulation means comprising at least one of:        -   a generator unit, and an electrode assembly coupled to the            generator unit and configured for creating inside at least            one portion of the microchannel an electric field which is            substantially collinear to the axis (X) of the microchannel,            wherein the generator unit is capable of generating the            electric field with such an amplitude and frequency that the            electric field causes the at least one packet to deform, or            the at least two packets to displace towards each other in            the microchannel,        -   at least one side channel with a first end in connection            with a portion of said microchannel and a second end in            connection with a delivery system suitable for delivering a            solution, with in particular a surfactant, able to alter the            interfacial tension between said at least two packets or            said at least one packet and the environment thereof, said            delivery system being configured to deliver said solution            into said microchannel at least during the passage of said            packet(s) in said portion of, the microchannel.

In other words, the manipulation of the packet(s) with the deviceaccording to the present invention may be carried out by generating anappropriate electric field, or by modifying the surface tension of aliquid packet, or by combining both techniques.

The interfacial tension may be modified, preferably decreased, by afactor of at least 20%, and more preferably 50%.

When two packets are introduced in the portion of the microchannel inwhich the electric field is applied to cause two packets to collapse,dipoles are created by the electric field in the packets, said dipolesbeing oriented substantially along the axis of the microchannel suchthat the packets attract each other and collapse.

The expression “substantially collinear” means that the average of thedirection of the electric field makes with the axis of the microchannelan angle smaller than 45°, for example smaller than 30°, preferablysmaller than 20°, and more preferably smaller than 10° or 5°.

The invention may be used for inducing the collapse of two packets, andfor example the coalescence of two droplets inside the microchannel.

The general physical phenomenon called “electrocoalescence” is disclosedin P. Atten, Electrostat, 30, 259 (1993). As electrophoretic forces, andin contrast with dielectrophoresis, electrocoalescence does not requitea field gradient.

When the packet is a cell, or contains several cells, the invention maybe used to induce electroporation of such cell or multiplicity of cells.

The invention may also be used to split a packet into several packets ofsmaller size, and for example to extract from a droplet one or severaldroplets of smaller size.

A micro-container according to the invention may have any size.Preferably, it has at least one dimension smaller than one millimeter.In an embodiment, said micro-container is a micro-capillary with adiameter smaller than 1 mm. For instance, at least one dimension incross-section of said capillary is preferably comprised between 100 μmand 1 mm. In an embodiment, at least one dimension of said capillary iscomprised between about 10 nm and 100 μm, preferable between 1 μm and100 μm. In another embodiment, said micro-container is amicro-fabricated microchannel with a thickness smaller than 1 mm. Insome embodiments, the thickness of said microchannel is preferablycomprised between about 100 μm and 1 mm. The thickness of saidmicrochannel may be comprised between about 10 nm and 100 μm, preferablybetween 1 μm and 100 μm.

In the present invention, “micro-container”, in particular“microchannel”, means a volume at least partly enclosed by solidsurfaces, said volume being small. Preferably, a micro-container, inparticular a microchannel, in the invention has a surface/volume ratiosubstantially greater than 1 mm⁻¹, preferably greater than 4 mm⁻¹, forexample greater than 10 min⁻¹, possibly greater than 1 μm⁻¹.Microchannels also encompass nanochannels.

In an embodiment, the microchannel is preferably elongated, i.e. thedimension along its axis is larger by a factor of 3, preferably by afactor of 10, for example by a factor of 100 or 1000, than along anyother direction perpendicular to said axis.

The axis of the microchannel may be rectilinear or not.

The microchannel may have a cross-section which is constant or not. Thesection may be for example circular, ellipsoidal, rectangular, square orwith a bowl shape.

In the present invention, “thickness” means the smallest inner distancein cross section between two opposite sides of the microchannel. As amatter of example, for a cylindrical microchannel having a circularcross section, the thickness is the diameter, For a slit-likemicrochannel having a rectangular cross section, the thickness is thelength of the small side of the rectangle.

The thickness of the microchannel can take any value between a few nmand a few mm. Preferably, the thickness is comprised between 1 μm and 1mm. Still preferably, the length of the microchannel along its axis, maybe chosen to be at least 10 times larger than the thickness. Themicrochannel may have a length chosen between 10 mm and several meters,for example between 1 cm and 50 cm.

Preferably, the portion of the microchannel in which the electric fieldis substantially collinear to the axis of the .microchannel has a lengthalong said axis at least as large as the thickness of the microchannel,and smaller than the total length of the microchannel. In a preferredembodiment of the invention, the length of said portion of themicrochannel is comprised between about 1 and about 100 times thethickness of said portion, and preferably between about 1 and about 10times the thickness of said portion.

The microchannel may be rigid or flexible and comprises for example atube made of a flexible non-electrically-conducting material.

The micro-container, in particular the microchannel, may be made of atleast one material selected among: fused silica glass, PDMS(polydimethylsiloxane), PMMA (polymethylmethacrylate), any kind ofelastomer or plastics, such as for example polyethylene, polyimide,epoxy, Teflon®, Parylene®, polystyrene, polyethylene terephtalate,fluoropolymer, polyester, cyclic olefin copolymer, non-conducting oxidesuch as, for example, glass, silicon dioxide, diamond, non-conductiveceramics, a silicone, an elastomer, a glassy material, a mineralmaterial, a ceramic, a polymer, a thermoplastic polymer, a thermocurableresin, a photocurable resin, a copolymer

In an exemplary embodiment of the invention, the microchannel has atleast one inlet port, and/or at least one outlet port. Optionally, atleast one of said ports can be connected to one or several reservoirs,to one or several pumps, to one or several detectors or sensors or toone or several sampling devices.

The microchannel may be part of a network of connected microchannels.

In a preferred embodiment of the invention, said electrode assembly iselectrically insulated from an inside surface of the microchannel, forexample by an insulating material. The insulating material may have athickness of at least 1 nm, for example at least 10 nm, preferably atleast 100 nm, most preferably of at least 1 μm, for example up toseveral tens or hundreds μm. Typically, thicker insulating layers may bepreferred for larger microchannels. This insulating material may be madeout of for example polymeric material, e.g. for example polyethylene,polyimide, epoxy, Teflon® Parylene®, PMMA, polystyrene, polyethyleneterephtalate, fluoropolymer, polyester, cyclic olefin copolymer, PDMS,non-conducting oxide such as, for example, glass, silicon dioxide,diamond, non-conductive ceramics.

Preferably, the electrode assembly comprises at least two electrodesaxially spaced along the axis of the microchannel by a distance kingenough for the electric field between the electrodes to be substantiallycollinear to the axis of the microchannel. Each electrode may besymmetric relative to the axis of the microchannel. Advantageously, atleast one of said electrodes comprises at least two equipotentialportions facing each other across the microchannel. At least one of saidelectrodes may be monolithic having for example a cylindrical surfacesurrounding the microchannel. In a variant, at least one of saidelectrodes is composite, i.e. made out of a plurality of pieces,comprising for example at least two substantially parallel equipotentialplates sandwiching the microchannel. The electrodes may also have ashape other than those described above.

This is different from the prior art as disclosed in patent applicationFR 2 794 039 or in Paik et al., Lab Chip, 3, 28-33 (2003) whereinmanipulation of the droplets is obtained by an opposite configuration,i.e. applying a potential difference between two planar electrodesfacing each other across the chamber in which the droplet to bemanipulated is contained.

Preferably, the electrodes are spaced by a gap having a length that isgreater than the thickness of the microchannel, preferably greater thantwice the thickness.

The generator unit comprises advantageously at least one of a currentand a voltage generator, configured to create a difference of potentialbetween said two electrodes, preferably an alternating potential.

Advantageously, the electrode assembly is configured so that in at leastone cross section of the microchannel the amplitude of the electricfield varies less than a factor 10, preferably less than a factor 5,better less than a factor 2, and preferably is substantially uniform, inparticular in the gap between the electrodes.

The electric field generated by the generator unit via the electrodeassembly may have any temporal profile, for example continuous, variableor alternating (AC), or a combination of such temporal profiles. Forinstance, the electric field may be an AC field with variable frequencyor root mean squared (RMS) amplitude, or a superposition of continuousand AC components.

By “AC field”, we mean any field periodic in time and with a zero timeaverage. Non limiting examples of AC fields according to the inventionare sinusoidal, square or sawtooth AC fields.

The generator unit is preferably capable of generating an AC electricfield with a frequency ranging from about 0.01 Hz to about 1 GHz,preferably from about 1 Hz to about 10 MHz.

The coalescence of droplets is efficiently achieved, for instance, withfrequencies between 100 Hz and 10 kHz. The aliquoting of at least onedroplet is preferably achieved at frequencies lower than 50 Hz.

The generator unit may be configured for delivering a RMS voltageranging between 1V and 30 kV, preferably between 60 V and 2 kV,depending on the nature of the packet, of the fluid surrounding thepacket, of the microchannel, and on the size of the device. The voltagemay increase as the size of the device increases. The RMS electric fieldinside the microchannel in the gap between the electrodes may range forinstance between 100 V/cm and 100 kV/cm, and preferably between 500 V/cmand 20 kV/cm.

The generator unit may be configured to deliver a voltage with at leastone of the amplitude and the frequency being time-related variable. Forexample, the amplitude and/or the frequency of the electric field may bemodified as two packets come into close contact.

Advantageously, the electrode assembly is housed in a support, thelatter having two separated support members assembled together via afixing element, each support member carrying one electrode of theelectrode assembly. The support may comprise at least one orifice forreceiving the microchannel.

When the microchannel is connected to a side channel, said side channelmay have a cross-section with dimensions comparable to those of themicrochannel. Preferably, the cross-section of the side channel issmaller than this of the microchannel. In a variant, the cross-sectionof the side channel is larger than this of the microchannel.

The delivery system associated with said side channel may comprisepressure control means or flow control means.

Preferably, said side channel and said deliver system are configured fordelivering in the microchannel a solution containing a surfactant.

The term “surfactant” means any species, molecules or combination ofmolecules capable of modifying the interfacial tension between twofluids. A surfactant may be for instance a tensioactive or anamphiphilic species.

The surfactant may be chosen to favor the formation of oil-in-wateremulsions.

If the packets are aqueous droplets suspended in a non-aqueous liquid,said surfactants are typically surfactants with a high HLB(Hydrophilic/Lipophilic Balance), for example with HLB values largerthan 15. Non-limiting examples of such surfactants are Sodium DodecylSulfate (SDS), oleic acid, and CTAB.

if the packets are oily droplets in an aqueous surrounding fluid, thesolution preferably contains at least one surfactant with a low HLB, forexample a HLB lower than 15, and preferably lower than 10.

Numerous surfactants able to reduce the interfacial tension between anoily phase and a water phase, or to favor droplet coalescence, arerecited e.g. in Emulsions, a fundamental and practical approach, J. S.Sjöblom Ed, Kluwer, Dordrecht (1992), or in P. Becher, Emulsions, Theoryand Practice, 2 Ed, R. E. Krieger Pub. Co, Malabar, Fla. (1985).

In an exemplary embodiment of the invention, for coalescing or splittingtwo droplets, the device comprising a first side channel connected tothe delivery system, the microchannel may be connected to a second sidechannel, preferably connected in regard or in close vicinity of thefirst side channel and configured for collecting packets formed by thecoalescence or splitting of original packets.

The microchannel may be made out of a wide variety of homogeneous orcomposite materials. in contrast with prior art disclosed in patent U.S.Pat. No. 6,294,063, in which packets are manipulated onto a reactionsurface configured to provide interaction sites for said packets, thewall of the microchannel according to the present invention may be madeof a material, or treated with a material, reducing the risk ofinteraction of the packets with the wall of the microchannel in thepresence of the embedding fluid. There may be no chemical interaction atall between the packets and the microchannel.

In an exemplary embodiment, the packet being a water-based droplet, theinterfacial tension between the droplet and the microchannel wall ismade larger than the interfacial tension between the droplet and thesurrounding fluid, by treating the microchannel wall and/or by includingin the droplet anti or in the fluid additives.

Numerous ways may be used to increase the surface tension between awater-based liquid and a surface. As an example, one may choose anaturally hydrophobic surface, such as fluorocarbon or polyethylene. Onemay also treat the surface with hydrophobic materials such as Teflon®AF, silane or fluorosilane.

The surface tension between water and hydrogenated oil-based fluids canbe decreased by the presence of surface-active molecules. Numerous suchsurface active molecules are known in the art, and we list here only afew as a matter of examples: surfactants such as Pluronic® andSymperionic®, Triton®, Tween®, Span® 80, Tergitol®, Sodium DodecylSulfate (SDS), oleic acid, methyl cellulose, hydroxyethyl and hydroxypropyl cellulose, or Coatex®. If the fluid is fluorinated, thenfluorinated surfactants, such as 1H,1H,2H,2H perfluorodecan-1-ol or1H,1H,2H,2H perfluorooctan-1-ol, are particularly suitable.

Fluorinated, water immiscible fluids may comprise at least one of thefollowing elements: partly or fully fluorinated alkanes, alcenes oralcynes, for instance perfluoroalkanes such as perfluomdecalin. In oneembodiment of the invention, the water-immiscible fluid is a mixture offluorinated molecules, such as the fluorinated solvents of the “Freon”family, or FC fluorosurfactant, such as FC40 or FC75 (commercialized by3M). Preferably none compound of a given molecular weight in saidmixture is representing more than 75% w/w (weight by weight) of themixture.

In an exemplary embodiment of the invention, the microchannel is filledwith a fluid surrounding at least one packet, said fluid may be anyliquid or gaseous fluid, provided it is not miscible in the packet orwith the wall of the microchannel.

The fluid surrounding the packet may be a liquid, for example awater-immiscible organic or inorganic liquid.

The fluid may he a fluorinated liquid or gas, and the droplet may be anorganic or hydroorganic liquid, optionally containing species.

In an exemplary embodiment of the invention, the packet and thesurrounding fluid have different conductivities and/or differentdielectric constants. For instance, the packet may have a conductivityhigher than the surrounding fluid conductivity.

In an exemplary embodiment of the invention, the packet is a droplet ofa first liquid suspended in an immiscible second liquid, said firstliquid being more electrically conductive than the first.

The droplet may be a water based droplet Said water based droplet maycontain any kind of natural, artificial, organic or inorganic speciessuch as, for example, biological molecules, proteins, protein complexes,enzymes, haptens, antigens, antibodies, aptamers, epitopes, nucleicacids, peptides, polysaccharides, glycopeptides, cells, cell aggregates,drugs, chemicals, latexes, living or dead organisms, viruses,organelles, liposomes vesicles, micelles, synthetic or natural polymers,nanoparticles, luminescent molecules, quantum dots, chemical reagents,buffers, surfactants, and any combination of such species.

In another exemplary embodiment of die invention, the packet is adroplet of a water-immiscible liquid, and the surrounding fluid is awater based solution.

The invention may allow for the manipulation of packets having a sizecomparable with the section of the microchannel.

As an exemplary embodiment, the area of the smallest section of saidpacket is at least equal to one half of the area of the section of themicrochannel at the location of a first electrode or at the location ofa second electrode, whichever is smaller.

The packet may be a spherical droplet with a diameter comparable withthe diameter of the microchannel, or an elongated droplet spanning thewhole section of the microchannel.

The device according to the present invention may be used to fusecolloids to form a chain, for instance.

The device may also be used for screening processing.

The invention also relates to, according to another of its aspects, amethod for displacing at least two packets towards each other in amicrochannel, in particular in order to collapse the at least twopackets, the microchannel having a longitudinal axis, said methodcomprising:

-   -   introducing the at least two packets in the microchannel,    -   generating an electric field within at least one portion of the        microchannel, at least when the packets are located within said        microchannel portion, said electric field being preferably        substantially collinear to the axis of the microchannel in said        portion and having an amplitude and a frequency chosen such as        to displace the two packets towards each other.

When the electric field is generated by at least two electrodes axiallyspaced along the axis of the microchannel, said electrodes beingseparated by a gap, the method may comprise:

-   -   before generating said electric field, positioning two packets        in the gap between the electrodes, said packets being in static        equilibrium,    -   generating said electric field.

In a variant, the packets for collapsing may be placed initially in aflowing stream such as to perform an in-flight operation of collapsing.

The method may thus comprise:

-   -   positioning two packets in the microchannel, at least one of        which being outside the gap between the electrodes,    -   displacing the packets towards the gap, for example via a        flowing stream in the microchannel,    -   generating said electric field at least when the packets are        located in the gap,

In an exemplary embodiment of the invention, at least one of saidpackets contains biological material, for example a cell or a cytoplasmnucleus. The method for collapsing at least two packets is particularlyadvantageous when the packets contain a biological membrane.

Said method may be carried out in order to form hybridoma or tomanipulate embryonic founder cells.

The invention also relates to, according to another of its aspects, amethod for displacing at least two packets towards each other in amicrochannel, in particular in order to collapse them, or for splittingat least one packet, the microchannel having an axis, said methodcomprising:

-   -   positioning the at least two packets or the at least one packet        in a portion of the microchannel,    -   delivering into said portion of the microchannel a solution of a        surfactant able to alter the interfacial tension between said at        least two packets or said at least one packet and the        environment thereof.

The invention also relates to, according to another of its aspects, amethod for splitting at least one packet in a microchannel having alongitudinal axis, said method comprising:

-   -   introducing the at least one packet in the microchannel,    -   generating an electric field within at least one portion of the        microchannel, at least when the at least one packet is located        within said at least one portion, said electric field being        preferably substantially collinear to the axis of the        microchannel in said portion and having an amplitude and a        frequency chosen such as to split the packet.

The invention also relates to, according to another of its aspects, amethod of monitoring the collapsing of at least two packets or splittingof at least one packet, the method comprising:

-   -   causing collapsing or splitting in a microchannel using the        microfluidic device as defined above,    -   detecting the collapsing or splitting, for example by using a        video device or by measuring an electric parameter such as an        electric resistance associated for example with at least one        substance contained in the microchannel.

The invention also relates to, according to another of its aspects, amethod for displacing at least one packet in a microchannel having anaxis, said method comprising:

-   -   introducing at least one packet in the microchannel,    -   generating an electric field within at least one portion of the        microchannel, at least when the at least one packet is located        within said portion, said electric field being preferably        collinear to the axis of the microchannel, such as to displace        the packet along the microchannel.

Said electric field may be continuous.

The operation of displacing at least one packet in the microchannel maybe performed independently from an operation of collapsing or splitting,

In a variant, said operation of displacing at least one packet in themicrochannel may be carried out in order to position appropriately saidat least one packet in the microchannel before performing, the operationof collapsing or splitting.

The invention relates to, according to another of its aspects, a methodfor performing at least one operation on at least one packet in amicro-container, in particular a microchannel, wherein saidmicro-container has at least one tubular portion defining an internalspace of the micro-container, wherein:

-   -   the tubular portion is made of a non internally coated bulk        fluorinated material, or    -   the tubular portion is made of a bulk non-fluorinated material        and is coated on all a circumference of an internal surface of        the tubular portion with a permanent layer, or        wherein the micro-container comprises a succession of at least        two tubular portions, a first tubular portion made of a bulk        fluorinated material and a second tubular portion made of a bulk        non-fluorinated material coated on all an inner circumference        with, a permanent layer, wherein said permanent layer is        preferably hydrophobic, and        wherein said micro-container is at least partially filled with a        carrier fluid immiscible with said packet and containing at        least one surfactant at a concentration large enough to decrease        the surface tension between said packet and said carrier fluid.

The expression “bulk material” means a monolithic material.

For instance, a bulk material according to the present invention isdifferent from an assembly of two portions made of the same material,for example of PDMS (polydimethylsiloxane).

A bulk material also differs from an assembly of two portions made ofdifferent materials, such as an assembly of a portion made of PDMSbonded to a portion made of glass or an assembly of a portion made ofborosilicate to a portion made of silicone.

In such known systems, the difference of chemical nature betweendifferent parts of the circumference of the micro-container ormicrochannel, tends to make interactions of the packet or of the carrierfluid with such parts different, and thus to provide poorer control overthe operations performed on said packet.

By “permanent layer”, we mean a layer which is not carried onto andremoved from the inner surface of the micro-container by the carrierfluid, as would be the case, typically, for a surface-active componentadded in the fluid, and in particular, surfactants such as SPAN, SOS,Pluronics, and the like. The use of a permanent layer is advantageoussince it is more robust, and it provides more freedom on the compositionof said carrier fluid.

In the method according to the present invention, for some applications,one may add to said carrier fluids such surfactants.

A tubular portion may have a circular or non-circular cross-section. Forinstance, the cross-section may be rectangular.

The cross-section of a tubular portion may vary or not along a length ofthe micro-container.

The permanent layer may comprise a material selected among: fused silicaglass, PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), anykind of elastomer or plastic, such as for example polyethylene,polyimide, epoxy, Teflon®, Parylene®, polystyrene, polyethyleneterephtalate, polyester, cyclic olefin copolymer, non-conducting oxidesuch as, for example, glass, silicon dioxide, diamond, non-conductiveceramics, a silicone, an elastomer, a glassy material, a mineralmaterial, a ceramic, a polymer, a thermoplastic polymer, a thermocurableresin, a photocurable resin, a copolymer, a silane, a fluorosilane, afluoropolymer.

The invention also relates to, according to another of its aspects, adevice for performing at least one chemical, physical or biologicaloperation on at least one packet embedded in a carrier fluid immisciblewith said packet, said device comprising at least a micro-containersurrounding said carrier fluid containing said packet, wherein the innersurface of said micro-container is fluorinated, and said carrier fluidcontains a surfactant at a ratio concentration of at least 0.1 cmc(critical micellar concentration).

The micro-container according to the invention can be of any shape. Itcan for instance be a rectangular section microchannel, a cylindricalmicrocapillary, a thin slab-like volume, or a cylindrical, pyramidal orrectangular microvial.

For some applications, the device according to the invention can gathermore than one, preferably more than 10 or more than 100 and up toseveral hundred thousands such micro-containers.

In an embodiment, the micro-container has at least one inlet port. Themicro-container may have at least one outlet port. Optionally theport(s) can he connected to one or several reservoirs, to one or severalpumps, or to one or several sampling devices.

Optionally, the micro-container can also be part of a network ofconnected microchannels and reservoirs.

The invention also relates to, according to another of its aspects, amethod for performing at least one operation on at least one packet in amicro-container, in particular a microchannel, wherein saidmicro-container has an inner tubular hydrophobic surface, wherein saidmicro-container is at least partially filled with a carrier fluidimmiscible with said packet and containing at least one surfactant at aconcentration large enough to decrease the surface tension between saidpacket and said carrier fluid.

Said operation may be at least one of displacing the at least onepacket, splitting the at least one packet, coalescing the at least onepacket with at least another packet, reacting the at least one packet.

The displacing may comprise circulating the carrier fluid in themicro-container.

The operation may be carried out in the absence of any electrical field.

The operation may comprise displacing the at least one packet from aninlet of the micro-container towards an outlet of the micro-container.

The operation may comprise exposing successively the at least one packetto at least two different physical and/or chemical conditions, inparticular to at least two different temperatures.

The invention also relates to, according to another of its aspects, amicrofluidic device comprising a micro-container, in particular amicrochannel, having an inner tubular surface, said device furthercomprising a tubular bulk hydrophobic portion forming an internal spaceof the micro-container wherein said bulk portion is coated with ahydrophobic layer.

In one embodiment, the bulk portion is made of a fluorinated material.In a variant, the bulk portion is made of a non-fluorinated material andcoated with a fluorinated layer.

The microfluidic device may comprise a succession of at least a firstand a second bulk portions, the first portion being made of afluorinated material, without coated layer, and the second portion beingmade of a non-fluorinated material and coated with a fluorinated layer.

In a variant, the microfluidic device may comprise two portions made ofTeflon® assembled together for forming a circumference of themicro-container.

The invention also relates to, according to another of its aspects, amethod for performing at least one operation on at least one packet in amicro-container, in particular in a microchannel, said micro-containerhaving an inner surface, wherein said micro-container is filled with acarrier fluid immiscible with said packet and containing at least onesurfactant, wherein the difference between the interfacial tensionbetween the packet and the inner wall of the micro-container and theinterfacial tension between the packet and the carrier fluid is at least26 mN/m, preferably at least 35 mN/m. Said difference may be comprisedbetween about 35 mN/m and about 45 mN/m.

In Tice et al., Langmuir 2003, 19, 9127-9133, it has been proposed thatthe transport of droplet by a carrier fluid in a microchannel isperformed without interaction with the microchannel wall, if theinterfacial tension between said droplet and said carrier fluid issmaller than the interfacial tension between the droplet and themicrochannel. In particular, these authors used water droplets,dissolved in a fluorocarbon containing a fluorosurfactant (interfacialtension 12-14 mN/m) in a PDMS microchannel, (interfacial tension 38mN/m).

In this case, the difference between the droplet/microchannel and thedroplet/fluid difference is 24-26 mN/M. Surprisingly, it has been foundthat in similar conditions, (water droplets in FC40 plus 1H,1H2H,2Hperfluorodecan-1-ol (interfacial tension between this fluid and water12-20 mN/m, see example 11 and FIG. 14) in a silicone capillary tube(interfacial tension of water to capillary tube: 38 mN/m), thiscondition (difference of surface tension positive and comprise between18 and 26 mN/m) is satisfied, however imperfect behavior of droplets,and contamination between droplets (see example 14) are still observed.

In contrast, the silicone is additionally treated with fluorosilane(interfacial tension with water: 55 mN/m), i.e. if the differencebetween the interfacial tension of the packet with regards to thesurface of the microchannel and the interfacial tension between thepacket and the fluid is increased to a value comprise between 35 and 45mN/m, no contamination is observed.

Also, when water droplets are transported in pure FC40 (water/PC 40interfacial tension: 51.8 mN/m) in a capillary tube of Teflon®(interfacial tension 55 mN/m), the condition stated in Tice et al. issatisfied, and nevertheless unsatisfying droplet transport is observed,and droplet breakage. In contrast, when the droplet/fluid interfacialtension is decreased to a value between 10 and 20 mN/m, by addition of0.5 to 3% 1H,1H,2H,2H perfluorodecan-1-ol, (difference between theinterfacial tension of the packet with regards to the surface of themicrochannel and the interfacial tension between the packet and thefluid to a value comprise between 35 and 45 mN/m), irregular dropletmotion and contamination can be suppressed.

In a preferred embodiment, the ratio concentration of the surfactant inthe carrier fluid is at least 0.1 cmc (critical micellar concentration),preferably at least 0.5, and more preferably 1 cmc.

Advantageously, the concentration of the surfactant in the carrier fluidis comprised between about 0.01% and about 10% w/w (weight by weight),preferably between about 0.1% and about 3%.

In a preferred embodiment, said surfactant is a fluorosurfactant, inparticular a fluoroalcohol. In a particular embodiment, thefluorosurfactant is 1H,1H,2H perfluorodecan-1-ol,

In one particular embodiment, said bulk portion is made of silicone. Forinstance, the microchannel is formed by a silicone capillary tube.

In one particular embodiment, the bulk portion is silanized. Forinstance, the inner surface of the bulk portion is treated with asilane.

In an embodiment, said silane is selected among monomethyl silanes,dimethylsilanes, trimethylsilanes, monochlorosilanes, dichlorosilanes,trichlorosilanes, and the like. In a preferred embodiment, said silaneis selected among monomethyl fluorosilanes, dimethylfluorosilanes,trimethylfluorosilanes, monochlorofluorosilanes, dichlorofluorosilanes,trichlorolluorosilanes, and the like. In a yet preferred embodiment,said fluorosilanes are perfluorosilanes. In an embodiment, said silaneshave tails with a skeleton involving at least 2 carbon atoms, preferably4 carbon atoms, yet preferably 8 carbon atoms, and yet preferable 12, 16carbon atoms or more. In an embodiment, said fluorosilane is selectedamong 1H,1H,2H,2Hperfluorooctyltrimethylsilane and1H,1H,2H,2Hperfluorodecyltriethoxysilane (Fluorochem). Said silane ispreferably dissolved in a water-free solvent such as ethanol, methanol,octane, DMF, and the like. In another embodiment, said silane isdeposited on the surface directly from gas phase, by blowing awater-free carrier gas over the surface of said silane, and then intothe microchannel.

In an embodiment, said silanization is performed under argon atmosphere.In another embodiment, said silanization is performed under airatmosphere. Preferably, said atmospheres are water-free, because watertend to hydrolyse the silane and prevent its grafting.

In another embodiment, said inner surface of said microchannel can beactivated prior to silanization, by one of several methods known fromthose skilled in the art. In one embodiment, said activation is a plasmaactivation. In preferred embodiment, said activation is performed byflowing an acidic solution in said microchannel. In one embodiment saidsolution is selected among chlorohydric acid, sulphuric acid, nitricacid, phosphoric acid, florhydric acid.

The invention also relates to, according to another of its aspects, to aconnector allowing: contamination free transport of at least one packetfrom at least one microchannel towards another microchannel, saidconnector being constituted by a bulk material having on all its innersurface a hydrophobic layer, said layer comprising one of a fluorinatedmaterial and a silanized material.

In a preferred embodiment, said bulk material is an elastomer.

In a further preferred embodiment, this connector is related to at leastone of its inlet(s) or outlet(s), to another microchannel made of adifferent material, preferably a non-elastomeric material.

In another embodiment, said connector has at least three orificesrelated by microchannels. Interestingly, also, this connector comprisesa portion that can be inserted into a pinch valve or a perisaltic pump,in order to control the flow of fluid inside said connector, between itsdifferent orifices.

Said connector can optionally be prepared by first preparing a moldedpiece, and then connecting to at least one of its orifices anelastomeric tubing, said elastomeric tubing also bearing on its innersurface a hydrophobic layer.

Preferably, and in contrast with most microchannels used in prior art tomanipulate packets in microfluidic systems, in the invention, thesection of the microchannel(s) is cylindrical: this avoids sharp angles,that tend to retain fluid or solid material, pin contact angles, andthus increase the risk of contamination between packets.

The invention also relates to, according to another of its aspects, amicro-container selected among:

-   -   a micro-container made of a bulk fluorinated material,    -   a micro-container made of a bulk non-fluorinated material having        a surface covered on all its circumference with a permanent        insulating layer,    -   and a microchannel comprising at least a portion made of a bulk        fluorinated material and at least a portion made of a bulk        non-fluorinated material, said portion made of a bulk        non-fluorinated material being covered on its all circumference        with a permanent insulating layer,        said micro-container being at least partially filled with a        carrier fluid immiscible with said packet and containing at        least one surfactant at concentration large enough to decrease        the surface tension between said packet and said carrier fluid.

The invention relates to, according to another of its aspects, amicrofluidic device comprising at least one micro-container, inparticular at least one microchannel, and a connector in communicationwith the micro-container, said connector being configured for connectingsaid micro-container to at least one of another micro-container, inparticular a microchannel, and an inlet or outlet port of the device,wherein said connector has an inner surface comprising at least onehydrophobic layer.

The invention also relates to, according to another of its aspects, aconnector configured for being mounted at one end of a capillary tubeforming a microchannel, wherein said connector has an inner surfacecomprising at least one hydrophobic layer.

The invention also relates to, according to another of its aspects, akit comprising:

-   -   a microfluidic device comprising a microchannel,    -   a connector as disclosed above, to be mounted on said        microfluidic device.

The invention also relates to, according to another of its aspects, akit for performing at least an operation on a packet comprising:

-   -   a micro-container, in particular a microchannel, having an inner        surface comprising at least one hydrophobic material,    -   a carrier fluid immiscible with said packet containing at least        one surfactant at a concentration large enough to decrease the        surface tension between said packet and said water-immiscible        fluid.

The invention also relates to an assembly comprises:

-   -   a connector as disclosed above.    -   at least one capillary tube connected to said connector.

The connector may be a T-connector.

Preferably, the capillary tube has a fluorinated or silanized innersurface.

The invention also relates to, according to another of its aspects, adevice for performing a PCR comprising:

-   -   to microchannel comprising a coil made of a capillary tube at        least partly filled of a fluorosolvent containing a surfactant,        the coil comprising a denaturing region, an annealing region and        an elongation region exposed to different temperatures.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood on reading the following detaileddescription of non-limiting embodiments, and on examining theaccompanying drawings, in which:

FIG. 1 is a diagrammatic partial view of a micro-fluidic deviceaccording to the invention,

FIG. 2 is a diagrammatic view in cross section of the device of FIG. 1,

FIG. 3 is a diagrammatic perspective view of a support member of thedevice of FIG. 2,

FIGS. 4A-4C and 5A-5C illustrate diagrammatically respectively threesteps of two coalescence operations according to the invention,

FIGS. 6A-6C illustrate diagrammatically and partially three steps of thealiquoting of a droplet according to the invention,

FIG. 7 is a diagrammatic view of an electric field distribution in aportion of the microchannel.

FIG. 8 is a diagrammatic partial view of a device according to a variantof the invention, and

FIGS. 9 to 13 illustrate diagrammatically and partially other variantsof the invention.

FIG. 14 plots the interfacial tension between fluorosurfactant1H,1H,2H,2H perfluorodecan-1-ol and fluorinated oil FC-40, as measuredin example 11,

FIG. 15 illustrates diagrammatically and partially a cycling PCR systemused in examples 12 and 13 to amplify DNA in segmented flow according tothe invention,

FIG. 16 is a gel electrophoresis of the product of amplification in atrain of droplets, as described in example 12,

FIG. 17 is a verification of the absence of contamination betweenneighboring droplets, as described in example 13,

FIGS. 18A-18E show droplet shapes in an untreated, unwashed siliconecapillary, with FC40 as a carrier fluid (a) Initial droplet, (b) Laterdroplet (>60^(th) droplet in the train): with fluorosurfactant solutionsat different concentrations (c) 0.15 wt % fluorosurfactant, (d) 0.5 wt %fluorosurfactant, (e) 3.0 wt % fluorosurfactant (examples 14A and 14B),

FIGS. 19A-19E show droplet shapes in silicon tubes silinazed in an Argonatmosphere, (a) 7.5 vol % silane, (b) 5 vol % silane, initial droplets,(c)'5 vol % silane, later droplets (>100^(th) droplet), (d) 1 vol %silane, (e) 0.25 vol % silane (example 14C),

FIGS. 20A-20E show droplet shapes in tubes silanized in air, (a) 10 vol% silane, 5 minute reaction, (b) 7.5 vol % silane, 5 minute reaction,later droplet shape (>110th droplet in train), (c) 5 vol % silane, 30minute reaction, HCl activation, (d) 5 vol % silane, 30 minute reaction,plasma activation (e) 2.5 vol % slime, 30 minute reaction (Example 14D),

FIGS. 21A-21B show droplet shapes in silanized silicon tubes with 0.5 wt% fluorosurfactant (a) 2.5 vol % silane, 30 minute reaction, 200th drop,(b) 5 vol % silane, 30 minute reaction, 200th drop (Example 14E), and

FIGS. 22 to 24 represent diagrammatically and partially a contaminationfree connector according to the invention.

1: FIRST EXEMPLARY EMBODIMENT OF THE INVENTION

FIG. 1 shows a microfluidic device 1 according to the invention, saiddevice comprising a microchannel 2 and an electrode assembly 3. Themicrochannel 2 has a longitudinal axis X and an internal cross sectionthat is circular.

The electrode assembly 3 comprises a pair of electrodes 4, eachelectrode 4 comprising a metal cylinder, for example aluminium. Thelength of each electrode 4 is for example 4 mm and the inner diameter1.5 mm and the outer diameter 1.9 mm.

The electrodes 4 are placed around the microchannel 2 and are spacedalong the axis X by a gap 6. The electrodes 4 are connected to agenerator unit 9 via connection elements 8 comprising electrical wires.

The electrodes 4 are housed in a support 10 comprising two supportmembers 11, each being a substantially rectangular parallelepiped madeof Plexiglas®, for example with a width of 24 mm, a height of 20 mm anda depth of 20 mm.

A first cylindrical hole 12, for example of diameter 1.9 mm, is drilledin each support member 11 along the axis X at the center of the supportmember 11 for holding the electrode 4. The first hole 12 extends from afront face 13 towards a rear face 14 of the support member 11, oppositeto the front face 13.

A second hole 17 is drilled perpendicular to the first hole 12 forreceiving a connection element 8 connecting the corresponding electrode4 to the generator unit 9.

Each support member 11 further comprises two holes 20 and 21 whose axesare parallel to the axis of hole 12 and configured for receivingrespectively a Teflon® screw 22 and a metal rod 23 in order to maintainthe support members 11 assembled with the holes 12 being collinear.

Each electrode 4 is mounted in the corresponding support member 11 suchthat the electrode 4 is flush with the front face 13, as illustrated inFIG. 2.

The front faces 13 of the support members 11 are spaced for example by alength of 2 mm defining a 2 mm gap 6 between the electrodes 4.

The generator unit 9 comprises for example a function generatorconnected to an amplifier such as to deliver sinusoidal voltages up to 2kV with frequencies up to 1 kHz. The generator unit 9 may also comprisea central processing unit such as a computer to programmably control thevoltage delivered to the electrodes 4.

FIG. 3 shows diagrammatically the orientation of electric field given bythe arrows together with the equipotential lines.

As one can see, the electric field is substantially collinear to theaxis X of the microchannel 2 and thus favors electrocoalescence andminimizes any effect of dielectrophoresis.

The device 1 may be mounted on an observation stage of a binocularmicroscope 30 connected to a CCD camera 31 and a video recorder 32, asillustrated on FIG. 1, thus enabling the monitoring of the collapsing oftwo packets or the splitting of a packet in the microchannel.

The device may be configured such that after the collapsing orsplitting, the packet(s) are drained off.

2: SECOND EXEMPLARY EMBODIMENT OF THE INVENTION

FIG. 8 shows an electrode assembly 3′ comprising two compositeelectrodes 35 each having a pair of substantially parallel equipotentialplates 36 facing each other and sandwiching a microchannel 2′ which hasa rectangular cross-section. Each pair of plates 36 is connected to arespective pole of the generator unit 9.

3: EXAMPLE OF AN OSCILLATION METHOD

In an embodiment, the droplet fluid is TBE 5× buffer (0.45 M Trisbase®,0.45 M boric acid and 0.01 M EDTA; Sigma®) dyed with 0.25 wt %bromophenol blue for observation in a carrier fluid of fluorinated oil(EC-40, 3M) with 0.5 wt % 1H,1H,2H,2H perfluorodecan-1-ol (Fluorochem®)added to prevent interactions with the wall of the microchannel 2. Thedroplet conductivity is 3 mS/cm and the carrier fluid conductivity is2.5.10⁻¹³ mS/cm. For droplet formation, the two fluids are layered in a1.5 ml Eppendorf® tube so that the bottom layer consists ofapproximately 0.6 ml of the carrier fluid (FC-40/1H,1H,2H,2Hperfluorodecan-1-ol) and the upper layer consists of approximately 0.6ml of the droplet fluid (TBE 5×/bromophenol blue).

The microchannel 2 is filled from a syringe pump (commercialised by KDScientific) using, for example a Hamilton® Gas-Tight 250 μl syringefilled with the carrier fluid. The excess fluid pumped into themicrochannel may be collected in a waste reservoir. After completelyfilling the capillary, the microchannel 2 is placed into the carrierfluid phase of the layered Eppendorf® tube. The pump is then aspiratedat a rate of 1 ml/hr. Droplets are formed by oscillating themicrochannel between the carrier phase and the droplet phase, eithermanually or by attaching the microchannel to a mechanical oscillator, atfor example approximately 2 Hz. Droplets formed by this method haveapproximately the same diameter as the channel.

4: EXEMPLARY METHOD FOR DISPLACING A DROPLET IN A MICROCHANNEL

The method may he carried out with any of the devices defined above.

A single droplet is formed by the oscillation method described above.Using the syringe pump, the droplet is aspirated into the gap 6 betweenthe electrodes 4. When the droplet has reached the section of the gapjust before the upstream electrode, the flow is stopped and the systemallowed to settle to equilibrium. A continuous voltage is then applied,with the positive voltage applied to the electrode 4 closest to thedroplet and with the farthest electrode grounded. The motion of thedroplet towards the grounded electrode can be recorded on video and thetime for a given displacement measured. The droplet only moves when itis between the electrodes 4 and stops when it is under the groundedelectrode.

5: EXAMPLE OF STATIC COALESCENCE

The static coalescence may be performed by any of the devices definedabove.

Droplets are formed by the oscillation method described above such thatthe spacing between the two droplets is larger than the gap 6 betweenthe electrodes 4. A first droplet is initially brought into the gap 6between the electrodes 4 using the syringe pump. When the dropletarrives at the upstream electrode, the flow is stopped. The droplet ismoved against the direction of the previously applied flow using theelectric field actuation described above until it reaches the downstreamelectrode. The flow is restarted until the first droplet returns to theupstream electrode. This procedure is repeated until a second dropletappears between the electrodes 4. The microchannel position in theelectrodes is then adjusted so that the second droplet is outside of thegap 6 between the electrodes. The first droplet is moved against thedirection of the previously applied flow until the gap between the twodroplets is for example 0.5 mm. The microchannel is then repositionedsuch that the midpoint between the two closest edges of the droplets iscentered between the two electrodes and the system is allowed to settleto equilibrium.

In the example depicted in FIGS. 4A to 4C, the first droplet 40 has adiameter of about 540 μm and the second droplet 41 has a diameter ofabout 560 μm. Upon applying a 2 kV, 1 kHz sinusoidal voltage to theelectrodes 4, initial droplet motion is steady, with the smaller droplet40 moving at a slightly higher velocity (FIGS. 4A and 4B). When thedroplets 40 and 41 come into close contact they rapidly accelerate anddrain the intervening film (FIG. 4C), which indicates that the device Ishould provide essentially instantaneous coalescence of droplets whichare initially close together, such as occurs after two droplets arrivesimultaneously at a T-junction.

6: EXAMPLE OF IN-FLIGHT COALESCENCE

The in-flight coalescence may be performed by any of the devices definedabove.

The first droplet 43 may have a diameter of about 580 μm and the seconddroplet may have a diameter of about 560 μm.

After positioning the droplets, the microchannel is displaced such thatboth droplets are outside the gap 6 between the electrodes 4. A 2 kV, 1kHz sinusoidal voltage is then applied to the 2 mm-spaced electrodes andkept on throughout the duration of the experiment. After applying theelectric field, the flow is started by aspirating with the syringe pumpat 50 μL/hr.

The droplet 42 enters the gap 6 between the electrodes 4 and moves at aconstant velocity in the absence of the droplet 43 (FIG. 5A). Theleading interface of the droplet 43 appears after 13 sec, but has noeffect on the droplet 42. Only when the trailing droplet is well insidethe gap 6 between the electrodes does the dipolar force manifest itselfThereafter, the coalescence time is essentially the same as in thestatic case (approximately 8 sec) for these widely separated droplets,but the dynamics are slightly different due to the flow. The dipolarforce is sufficiently strong to stop the droplet 42 (FIG. 5B), whereuponthe droplet 43 moves toward it at a constant rate, closing the distanceat essentially the same rate as in the static case. Once the dropletsare close together, the strong dipolar force rapidly drains theintervening fluid and coalescence is achieved (FIG. 5C).

7: EXAMPLE OF DROPLET SPLITTING

A single large droplet 46 is formed by oscillating the interface asdescribed above but at a lower frequency. The droplet 46 is brought intothe gap 6 between the electrodes, by aspirating with the syringe pump(FIG. 6A). The drop depicted in the present embodiment is an ellipsoidof revolution with a 2.5 mm long axis. Upon applying a 2 kV, 0.1 Hzsquare tension, the drop 46 splits into two smaller, stable drops 47(FIGS. 6B and 6C) that are ejected from the gap 6.

Typical operating conditions for achieving a clean droplet splitting,that is to say one big droplet splitting into two smaller and stableones without formation of any satellite drops may consist in a squarevoltage with a frequency between 0.1 and 1 Hz and an amplitude between 1kV and 2 kV. Under such condition, the droplet may break in less than 1minute. The lowest the voltage applied, the “cleanest” the splitting butthe longer it may take. The droplet length may be about the length ofgap 6.

8: OTHER EXEMPLARY EMBODIMENTS OF THE INVENTION

As illustrated in FIGS. 9 and 10, the microchannel portion 50 betweenelectrodes 4 may form a T-intersection with a transverse channel 51.After coalescence of the droplets caused by the electric field betweenthe electrodes 4, the resulting droplet 52 may be driven in thetransverse channel 51, for example by using a syringe pump connected tothe transverse channel 51.

As illustrated in FIGS. 11 and 12, the droplet splitting may beperformed by extracting a droplet 53 from a relatively large mass offluid 54 by applying the electric field between the two electrodes 4.

Thus droplets 53 can be formed when, desired, for example byprogrammably controlling the electrodes 4.

9: ANOTHER EXEMPLARY EMBODIMENT OF THE INVENTION

FIG. 13 shows a device 60 according to the invention, said device 60comprising a microcbannel 61 connected in a portion 62 to first andsecond side channels 63 and 64.

Portion 62 may for instance be situated substantially at the middle ofthe microchannel.

In the present embodiment, the microchannel 61 may have a thickness ofabout 100 μm and a width of about 300 μm and the side channel 63 athickness of about 100 μm and a width of about 50 μm.

The side channel 63 is connected to a delivery system 66 comprising asyringe pump having a reservoir 67 containing a solution of surfactantof oleic acid and SDS in hexadecane, at a concentration superior to thecritical micellar concentration.

The microchannel 61 is filled with a solution containing hexadecanecontaining SPAN®80 at a concentration adjusted to avoid interaction ofaqueous droplets with the microchannel walls.

The side channel 64 is connected to a springe pump 68 in aspiration modeconfigured to aspirate the solution from the microchannel 61.

Two droplets 70 of a 5×TBE Buffer are introduced and displaced in themicrochannel 61 by its both ends. The aspiration of droplets 70 by bothends is synchronized so that the droplets 70 arrive from both sides atthe same time at the portion 62. When the droplets 70 are in the portion62, a solution of surfactant contained in the reservoir 67 is deliveredby the delivery system 66 into the portion 62 of the microchannel with apredetermined flow rate such as droplets 70 coalesce. The optimal flowrate may be determined by progressively increasing the flow untildroplets coalesce at each collision, which sets the optimal flow rate.

In another embodiment, the solution of surfactant may be delivered inpulses synchronized with the arrival of the pair of droplets at theconnection portion 62.

The resulting droplet 71 is collected in syringe pump 68 for furtheruse, or e.g. transferred to another microchannel for detection.

10: EXAMPLE OF FORMATION AND TRANSPORTATION OF REGULAR ARRAYS OF WATERDROPLETS IN A FLUORINATED OIL CONTAINED IN A FLUOROPOLYMER CAPILLARYTUBE

Trains of droplets are created by using a Y-connector (UpchurchScientific) connected to an electro-pinch valve (NResearch, CaldwellN.J.). One entry to the Y-connector was filled with TBE 5× buffer (0.45MTrisbase, 0.45M boric acid, and 0.01M EDTA, Sigma) dyed with 0.25 wt %bromophenol blue for ease of observation. The other side of theY-connector and the test capillary (PFA, i.d. 800 μm, UpchurchScientific) were primed with either the bulk fluorinated oil FC-40 (3M)or FC-40 containing various amounts of a fluoro alcohol surfactant(1H,1H,2H,2H perfluorodecan-1-ol, Fluorochem). Droplet trains werecreated by cycling the electrovalve with a LabView program whileaspirating with a computer-controlled Harvard milliliter module syringepump. A typical cycle consists of 6 seconds of aspiration from the TBEline and 8 seconds of aspiration from the FC-40 line. The droplets wereobserved with a binocular microscope (Olympus) and recorded using a CCDcamera (Hitachi) and WinTV.

The stability of droplet trains in the fluidic system is first tested,in previous work on segmented flow PCR, cross-contamination betweendroplets was attributed to droplet instability and the formation ofsmall satellite droplets. We not only wanted to look for dropletbreakage in individual droplets, but also to observe the overallstability of droplet trains containing several hundred droplets. Thestability of such trains is essential for high-throughput applicationsof our technique.

When the droplets are entrained in pure FC-40, it has been observed thatthey occasionally stick to the walls. This is unexpected, since both thewalls and the carrier fluid are fluorinated, and it has been expectedthat the walls will be strongly wetted by the FC-40. The sticking hasbeen attributed to imperfections (either roughness or chemicalinhomogeneities) in the capillary walls, which would be expected in bulkcapillaries manufactured by an extrusion process. The layer of FC-40between the droplets and the capillary wall is very thin, and smallperturbations to the wall surface could disrupt the lubrication flow. Inany event, once one droplet becomes entrained on the wall, eventemporarily, the train as a whole loses its stability. The trailingdroplet collides with the entrained droplet and exchanges fluid, theentrained droplet is released from the wall, and the trailing dropletbecomes entrained. This process proceeds ad infinitum and would becatastrophic in any PCR application.

The fluoroalcohol surfactant is then added to FC-40 in the range of0.5-3.0 wt %. Upon making trains containing over 200 droplets, notransient pinning to the walls, satellite droplet formation, orinstabilities in the droplet train are observed.

11: MEASUREMENT OF INTERFACIAL TENSION BETWEEN A WATER DROPLET AND ASOLVENT CONTAINING FLUOROSURFACTANT

Interfacial tension measurements were made using a homemade drop-volumetensiometer. The FC-40/surfactant drop is dispensed into a reservoir ofTBE 5× buffer front a 0.8 mm 1D Teflon® capillary tube. Using a 50 μLHamilton gastight syringe and a Hamilton PSD/2 syringe pump at maximumresolution, the drop volume could be increased in 25 nL increments witharbitrary waiting times between steps. To allow for equilibration of thesurfactant, we typically waited 30 seconds between steps. The tensionmeasurements are the average of at least 15 different drops, correctedfor wetting of the Teflon® tip.

12: EXAMPLE OF PCR AMPLIFICATION OF DNA IN A DEVICE ACCORDING TO THEINVENTION, INVOLVING A CHANNEL MADE OF BULK FLUOROPOLYMER

A PCR device 80 is depicted in FIG. 15, said device 80 comprising a 4cm, diameter copper cylinder 81 machined into three pieces correspondingto the denaturing 82, annealing 83, and elongation 84 regions. Theelongation region 84 is twice the size of the other two regions, thusoccupying half the cylinder 81. The three regions are isolated one fromanother by polycarbonate sheets 85, which are affixed between the piecesof the copper cylinder. The two ends of the heater are capped with apolycarbonate cylinder to provide structural stability while maintainingisolation between the two zones. Three small holes 88 per quartercylinder are drilled through the entire device to provide vents for aircooling. Two small holes 89 (for the thermocouples) and one largercentral hole 90 (for the heater) are drilled partially through thecylinder in each temperature region. The beating and thermocouple holesare not open to the air-cooling side of the heater. Each region iscovered with a thin layer of aluminum foil in contact with therespective region of the copper cylinder to provide heating from above.The foil layers are covered by a polycarbonate shell and a layer ofcotton, thereby insulating the cylinder and providing a uniformtemperature across the capillary. A small turbine blows ambient airthrough the three ventilation holes per quarter-cylinder, allowing forbetter temperature control and uniformity.

Each region includes a resistance heater and two Pt-100 thermocouples.The resistance heaters are located in the center of their respectivezone, while the thermocouples are located near the interface betweenzones. The heating elements and thermocouples are connected to customelectronics. The thermocouples communicate with a custom PID programcontrol written in LabView via a Keithley 2701 multimeter. Thetemperature of each zone can be set arbitrarily. For the experimentsdiscussed here, it has been used a denaturing temperature of 94° C., anannealing temperature of 55.5° C. and an elongation temperature of 72°C. With our design, a temperature difference of ±0.2° C. across eachzone is achieved.

A 4.5 meter long transparent PFA capillary tube 92 (i.d. 800 μm,Upchurch Scientific) enters the cylinder through a groove in thedenaturing region, providing an initial denaturation step ofapproximately 1 minute. The capillary is then wound 35 times around thecylinder, corresponding to 35 PCR cycles. The capillary exits the heaterthrough a hole in the extension segment, providing approximately 30seconds of additional extension on the 35^(th) cycle.

PCR Amplification: The template is a 2823 base pair DNA fragment ofLitmus 28i (New England Biolabs). This fragment is amplified on 572 basepairs from base 2008 to base 2580 using Eurogentec primers (lower primer5′-CGC-ATT-GCG-GTA-TCT-AGA-ACC-GGT-GAC-GTC-3′, upper primer5′-AGC-TTG-GAG-CBA-ACG-ACC-3″ Eurogentech Oligold). A 50 μL PCR mix isprepared using the Ready Mix Taq reaction mixture (Sigma) according tothe manufacturer's specifications with the maximum concentration oftemplate and primers.

The carrier fluid is a bulk fluorinated oil FC-40 (3M) containing0.5%-1.0% wt. Fluoroalcohol surfactant (1H,1H,2H,2H perfluorodecan-1-ol,Fluorochem). The surfactant prevents the transient adsorption ofdroplets to the capillary walls. The 2 μL, aqueous droplets are injectedinto the inlet by aspirating from the capillary outlet using a HamiltonPSD/2 pump and a 100 μL Hamilton gastight syringe. The drops areseparated one from another by a 5 μL FC-40 spacer. After injecting thedesired number of droplets, the outlet is disconnected from the Hamiltonpump and the inlet is then connected to a computer-controlled Harvardmilliliter-module pump with a 5 mL Hamilton gastight syringe. Thedroplets are circulated at 0.1 cm/s.

The droplets are collected at the outlet and analyzed by gelelectrophoresis on a 1 wt. % agarose gel in 0.5× TAE butler. A controlamplification sample is made by amplifying the remaining volume of the50 μL PCR mix in a classic PCR thermal cycler (Perkin Elmer) with acycle of 1 minute at 94° C., followed by 35 cycles of 94° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. This mimics thecycling in our continuous-flow PCR, although the lag time for heatingand cooling the classic cycler means that the total amplification isapproximately twice as long as our flow device. A 2 μL aliquot of theamplified control system is used for the gel electrophoresis.

It has been made a train of five droplets, each one containing the PCRmix and template. The result of this successful amplification in alldroplets is depicted in FIG. 16. Lane 1: 1 kbp DNA ladder (New EnglandBiolabs), Lane 2: 2 μl control sample of mix with DNA, Lanes 3: Droplet1, Lane 4: Droplet 2, Lane 5: Droplet 3, Lane 6: Droplet 4, Lane 7:Droplet 5.

The degree of amplification in the device according to the invention(lanes 3 to 7) is comparable to that obtained in the conventionalthermal cycler (lane 2).

EXAMPLE 13 Study of Contamination Between Droplets in a Channel Made ofBulk Fluoropolymer

The system has been tested for cross contamination between droplets. Allconditions are identical to Example 12, except that two separate PCRmixes are made; a first mix contains the template, primers and Ready Mixreaction mixture and the second mix is identical except that it does nothave any template. Five droplets are aspirated, but only the thirddroplet contains the template, in order to avoid contamination from thetip itself, the latter has been washed it in distilled water betweeneach droplet injection. FIG. 17 shows the gel electrophoresis resultfrom this experiment. Lane 1: 1 kbp DNA ladder (New England Biolabs),Lane 2: 5 μL, control sample of mix without DNA, Lane 3: 2 μL controlsample of mix with DNA, Lane 4: Droplet 1 (no template), Lane 5: Droplet2 (no template), Lane 6: Droplet 3 (with template), Lane 7: Droplet 4(no template), Lane 8: Droplet 5 (no template). There is no observablecontamination between different droplets—the only droplet exhibiting anyDNA amplification is the third droplet, which contained the DNA.

EXAMPLE 14 Examples of Droplet Transport in a Channel Made of aNon-Fluorinated Material With and Without Coating With a Layer ofFluorinated Material

In this series of example, the microchannel is made of Silicon tubes(inner diameter 0.8 mm) commercialized by Cole Parmer. The carrier fluidis FC-40 (3 M), and the droplets are made of the aqueous buffer TBE 5×.In all cases, a train of droplets is created following the same protocolas described in Example 13, and the shape and migration of droplets inthe tube is directly observed and photographed with a binocular and CCDcamera.

14A: Droplets of TBE in Pure FC40 in Untreated Silicone Tube

In some instances, the droplets appeared to have a spheroidal shape(FIG. 18a ) which indicates that the walls were not wetting. However,after forming a droplet train with several hundred droplets, the wallsbecame wetting at some point during the train (FIG. 18b ). In someinstances, the first droplets looked like FIG. 1b . It has been presumedthat the initially non-wetting behavior observed in some instances isdue to a residual product from the manufacturing process. As manydroplets are moved through the system, this unknown product is removedfrom the walls. To test this hypothesis, the tubes are washed with 5volumes of distilled, purified water. Afterwards, the first dropletsalways have a behavior like FIG. 18b . For all subsequent experiments,the tubes are always with 5 volumes of distilled, purified water toremove any variations in the it initial conditions.

14B: Droplets of TBE in FC40 with Fluorosurfactant Added, in UntreatedSilicone Tube

The behavior of the droplets is tested with the addition of various wt %or a fluorosurfactant, 1H,1H,2H,2H perfluorodecan-1-ol, Fluorochem. Thesurfactant reduces the interfacial tension between the droplets andFC-40 but does not affect the solid-liquid tension. As a result, thedroplets are destabilized and break into many small droplets (FIGS. 18c, d, e) The nature of the breakup depends on the surfactantconcentration but even at extremely low levels of fluorosurfactant thedroplets are still unstable.

14C: Droplets of TBE in FC40 Without Fluorosurfactant Added, in aSilicone Tube.Treated by Silanization in Argon Atmosphere

The tubes were silanized while isolated in an Argon atmosphere. A smallquantity of 1N HCl (Sigma) was heated to approximately 60 C on ahotplate. One end of a cleaned silicon tube was connected to a syringeof at least double the volume of the tube and the other end was placedin the warm HCl solution. The HCl was aspirated into the tube until thesyringe was partially filled. The HCl was left in the tubes for 5minutes, during which we occasionally oscillated the syringe pump toprovide local mixing. The HCl was then evacuated from the tube and thetube was dried with a flow of Argon. We then connected a new syringe toone end of the tube and placed the other end of the tube in a solutionof fluorosilane and spectroscopic grade methanol (Sigma). Thefluorosilane is usually 1H,1H,2H,2H,-perfluorooctyltrimethylsilane(Fluorochem). 1H,1H,2H,2H-perfluorodecyltriethoxysilane (Fluorochem) hasalso been tested and essentially the same results are achieved. All theresults shown here are for 1H,1H,2H,2H,-perfluorooctyltrimethylsilane(Fluorochem), which we chose to use because it is less expensive. Thesilane solution is aspirated into the tube and left for 5 minutes,during which we occasionally oscillated the syringe pump to providelocal mixing. The silane solution was evacuated from the tube and thetube was dried with a flow of Argon. The dried tube was then placed inan oven at 110 C for approximately 20 minutes to fix the slimes. Thetube was then washed with several volumes of methanol and FC-40 beforeperforming the droplet test.

The results of droplets in Argon-silanized tribes are shown in FIG. 19.For vol % greater than 7.5, we observed no pinning, for trains includingat least 200 droplets. At 5 vol %, the droplets appeared to be initiallynonwetting, but eventually pinned to the wall. For lower vol %, thedroplets always wetted the wall. We concluded that 7.5 vol %fluorosilane is necessary to form a stable, nonwetting surface when thereaction is performed under Argon.

14D: Droplets of TBE in FC40 Without Fluorosurfactant Added, in aSilicone Tube.Treated by Silanization in Air Atmosphere

To simplify the silanization procedure, the tubes are silanized in air.In order to better preserve the pure silane, the silanelmethanol mixtureis first performed in Argon but the remainder of the reaction is thenperformed in a hood using essentially the same protocol as above. Thevol % of silane and the time that the silane was allowed to react withthe tube (reaction time) are varied. In one instance, the HCl activationstep is replaced with plasma activation.

For a 10 vol % silane and 5 minute reaction time, the silanization inair was indistinguishable from the case in Argon (FIG. 20a ). At 7.5 vol% silane and a 5 minute reaction, the droplets initially appearednonwetting but eventually wet the walls (FIG. 20b ). It has been foundthat at 5 vol % silane the droplets still wet the walls, but when thereaction time is increased to 30 minutes, the droplets were nonwettingthroughout the entire train (>200 droplets, FIG. 20c ). The sameparameters (5 vol % silane, 30 minute reaction) are tested but the tubeis placed in plasma for 1 minute rather than filling it with HCl. As inFIG. 20d , the plasma activation did not result in a nonwetting surface.Since the plasma must diffuse through the tube for activation, it isless efficient than the liquid activation by HCl, which can he pumpedthrough the tube. It has been tested further lowering the silaneconcentration to 2.5 vol % silane while retaining the 30 minute reactiontime. As in FIG. 20e , the walls are slightly wetting.

The silanization in air probably produces a less uniform coating on thesurface than in Argon, since the water in air competes with the silanefor the activated surface sites. In essence, the air silanization doesnot reduce the water-solid tension as much as the Argon silanization.However, the air protocol is much simpler to perform and more amenableto automation.

14E: Droplets of TBE in FC40 with Fluorosurfactant Added, in a SiliconeTube.Treated by Silanization in Air Atmosphere

It has been checked if fluorosurfactants, which strongly reduce theEC-40/water interfacial tension, are sufficient to overcome thenonuniformities (and concomitantly lower water-solid tension) thatarises from the air-silanized surface. As indicated in FIG. 21, a smallconcentration of surfactant (0.5 wt %) is sufficient to prevent wettingat both 2.5 vol % silane and 5 vol % silage, even after forming a trainof 200 drops.

EXAMPLE 15 Verification by Quantitative PCR, of Contamination by DNABetween Droplets Transported in Devices According to the InventionPrepared in Example 14

To test whether unpinned droplet shapes do not lead to contamination, aset of experiments has been performed using quantitative PCR to make asensitive test of the DNA concentrations in different droplets.

The tubes were prepared according to one of these protocols:

-   -   1. No Silane: Tubes were washed with several volumes of        distilled water only. Carrier fluid is FC-40 (3M), prepared        according to Example 14A.    -   2. Silane: Tubes were washed and then silanized in air according        to the protocol in Example 14D with 5 vol % silane and 30 minute        reaction time. The carrier fluid is FC-40.    -   3. Silane+Surfactant: Tubes were washed and then silanized in        air according to the protocol in Example 14E. The carrier fluid        is FC-40 with 0.5 wt % 1H,1H,2H,2H perfluorodecan-1-ol.    -   4. Bulk fluoropolymer+Surfactant. A train of droplets is        prepared according to example 13: Teflon capillaries were used        as supplied by the manufacturer. The carrier Bind is FC-40 with        0.5 wt % 1H,1H,2H,2H perfluorodecan-1-ol,

One end of the tube was connected to a Y-connector, and the outlets ofthe Y-connector were selected using au electro pinch valve. One outletgoes to a Harvard millilitre module syringe pump and a 5 ml Hamiltongastight syringe and die second outlet goes to a Hamilton PSD/2 syringepump with a 100 μl Hamilton gastight syringe. The Hamilton PSD/2 wasused to make all of the droplets (by aspiration) or dispense thedroplets from the tube (by pumping). The Hamilton millilitre module wasused for droplet oscillation inside the tube. Prior to each experiment,the capillary was filled completely with the carrier fluid and the openend was placed in a reservoir of carrier fluid.

The droplets are mixtures of Taqman PCR mix for quantitative PCRcontaining Gold Tact polymerase enzyme (Applied Biosystems), qPCR CoreReagent Kit (Eurogentec), specific and a primers, fluorescent probe(3′-ATCTGCTGCATCTGCTTGGAGCCCA-5′, Applied Biosystems). “Mix” samplescontained all of the components for PCR except for the template. The“DNA” samples contain cDNA isolated from cell line A549 at aconcentration of 6.25 μg/μl. The fragment is amplified on 149 bycorresponding to the RPLPO gene using Proligo primers (upper primer3″-GGCGACCTGGAAGTCCAACT-5′; lower primer 3′-CCATCAGCACCACAGCCTTC-5′). Wemade two reservoirs for each experiment, one reservoir with sufficientmix for each control droplet (typically 30 μl) and a second reservoirwith sufficient mix and template for the cDNA droplets (typically 22μl).

It has been tested for contamination during injection by the followingprocedure. 2 μl has been aspirated from the DNA reservoir and then 4 μlfrom the carrier fluid reservoir. The tip was then washed by dipping itin a reservoir of distilled water and drying with a ChemWipe. Thesubsequent 5 drops were formed by aspirating 2 μl from the mix reservoirand 4 μl from the carrier fluid reservoir. After all of the dropletswere formed, we reversed the procedure and collected each droplet in aseparate Eppendorf tube. The eppendorf tubes were stored at −80 C priorto the quantitative PCR.

The contents of each drop were analyzed by quantitative PCR on a Taqman7700 qPCR machine (Applied Biosystems), In these experiments, a value of35 indicates that there was no detectable amount of cDNA in the droplets(i.e. 35 cycles of amplification without a fluorescence signal above thenoise threshold), and each integer increment corresponds to a halving inthe mass of cDNA,

Table 1 presents the results from the inlet contamination experiment.There was significant contamination in the untreated capillary, as wouldbe expected from the droplet shape. There was also contamination in thesilanized capillary. However, there was no contamination in the mixreservoir, so we could conclude that the contamination occurs fromdroplet transport inside the capillary. For the silanized capillary withfluorosurfactant we observed some contamination in the first washdroplet and equivalent contamination in the mix reservoir. This led usto believe that the contamination occurred at the tip during transfer,and that more vigorous washing, of the tip after the aspiration of theDNA droplet should be sufficient to eliminate contamination at the inletusing a silanized capillary and fluorosurfactant. There was nocontamination using the Teflon tip.

We then tested the ability of the fluorosurfactant and silanizedcapillary to prevent contamination while the droplets were in transit.Using essentially the same aspiration procedure as above with thefluorosurfactant and a silanized capillary, we first made two mixdroplets, then a cDNA drop, and then two mix droplets. The differencebetween the present injection protocol and the one above is that we nowwashed the tip in two separate distilled water reservoirs afterinjecting the DNA drop, with the aim of reducing the contamination atthe tip. At the end of the procedure we had a 2 cDNA drop with two 2 μlmix droplets on either side of it (Control 1 and 2 leading, Control 3and 4 trailing), where each droplet was spaced by 4 μl of the carrierfluid.

We then aspirated using the Harvard millilitre module so that thedroplets were in a straight section of the capillary positioned below anOlympus binocular microscope. Using computer control, we oscillated theHarvard millilitre module so that it pulled the droplets 5 cm in thecapillary at an average velocity of 1 mm/sec, and then pushed thedroplets 5 cm at an average velocity of 1 mm/sec, so that a single cycleresulted in no net displacement of the droplets. We performed 50 suchcycles, so that the total distance travelled by the droplets (5 m) iscomparable to that required in our continuous flow PCR machine. Byoscillating the droplets, rather than pushing them at a constant ratethrough a 5 m capillary, we simulate the conditions in a high throughputoperation. We occasionally observed the droplets with the microscope toensure that they were not wetting the walls.

After completing the 50 cycles, we collected the droplets in individualeppindorf capillaries. After ejecting each droplet, we aspirated a 2 μlwash droplet of distilled water to clean the tip. The wash droplets werealso collected. All of the droplets and the mix reservoir were analyzedby quantitative PCR as above.

Table 2 presents the results of the quantitative PCR. There was nodetectable contamination in any of the control drops. Moreover, therewas no detectable contamination in the wash droplets. There was somecontamination in the mix reservoir, but this did not lead tocontamination in any of the control droplets. We conclude that thecombination of the fluorosurfactant and a silanized capillary should besufficient to prevent contamination in high throughput applications.

We performed essentially the same oscillation experiment using a Tefloncapillary and fluorosurfactant. The only difference is that in thisexperiment we did not wash the tip when collecting the droplets or usethe extra wash droplets.

The results with a Teflon tube and fluorosurfactant are presented inTable 3. As in the inlet contamination test, there is no contaminationin the mix, indicating that the washing is sufficient to remove the DNAfrom the tip. There is some contamination in drop 2. We believe thatthis contamination is due to not washing the tip when collecting thedroplets. Drop 2 exits the system immediately after the cDNA drop. It ispossible that a small pan of the cDNA drop may have become entrained onan imperfection in the tip surface, which would then be transferred toDrop 2. An automated droplet collection or in-line detection procedureshould eliminate this source of contamination.

TABLE 1 Quantitative PCR results for contamination at the inlet. 35.00corresponds to no detectable cDNA, and each integer decrementcorresponds to a doubling in the relative cDNA mass. Silane + Teflon +No Silane Silane Surfactant Surfactant Q PCR Q PCR Q PCR Q PCR DropletValue Droplet Value Droplet Value Droplet Value cDNA 19.90 cDNA 20.32cDNA 20.29 cDNA 19.74 Wash 1 32.99 Wash 1 32.18 Wash 1 32.89 Wash 135.00 Wash 2 35.00 Wash 2 33.66 Wash 2 34.36 Wash 2 35.00 Wash 3 32.15Wash 3 34.91 Wash 3 34.77 Wash 3 35.00 Wash 4 32.54 Wash 4 35.00 Wash 435.00 Wash 4 35.00 Wash 5 35.00 Wash 5 34.92 Wash 5 35.00 Wash 5 35.00Mix Reservoir 33.15 Mix Reservoir 35.00 Mix Reservoir 33.13 MixReservoir 35.00

TABLE 2 Quantitative PCR results for contamination during cycling in asilanized capillary with fluorosurfactant. 35.00 corresponds to nodetectable cDNA, and each integer decrement corresponds to a doubling inthe relative cDNA mass. Droplet Q PCR Value Control 1 35.00 Control 235.00 cDNA 20.17 Control 3 35.00 Control 4 35.00 Wash 1 35.00 Wash 235.00 Wash 3 35.00 Wash 4 35.00 Wash 5 35.00 Mix Reservoir 33.90

TABLE 3 Quantitative PCR results for contamination during cycling in aTeflon capillary with fluorosurfactant. 35.00 corresponds to nodetectable cDNA, and each integer decrement corresponds to a doubling inthe relative cDNA mass. Droplet Q PCR Value Control 1 35.00 Control 230.19 cDNA 19.83 Control 3 35.00 Control 4 35.00 Mix Reservoir 35.00

EXAMPLE 16 Design and Fabrication of a Contamination Free T-Connector

This example illustrates the conception of a PDMS T-connector 100 with avery low dead volume allowing to connect without contamination threedifferent entries compatible with the use of pinch valves.

The connector 100 is made in PDMS (KODAK SYLGARD 184) with a 1:10proportion of curing agent. The mould 101 used for manufacturing theconnector 100 is a PMMA parallepiped (inner length and width 9 mm, innerheight 8 mm). 800 μm inner diameter holes 102 are drilled on the centerof three faces 103 of the parallepiped, A first 5 μm piece 104 ofTeflon' capillary 105 (outer diameter 800 μm) is introduced inside theend of a 3 cm long second Teflon capillary (outer diameter 1.5 mm, innerdiameter 800 μm) and sticks out on 1.5 mm. Three pieces are made thisway and are introduced and maintained inside the parallepiped so as toform a T with the smaller tubing facing each other (see FIG. 22). ThePDMS 106 is then degassed for 20 minutes, poured in the so constructedmould and put in a 65° C. oven for three hours.

After three hours, the Teflon® pieces 104 and 105 are taken off bygently pulling on them, and the obtained T-connector is taken out of themould 101. The T-connector 100 has three ports 108, each comprisingcoaxial cylindrical hollow portions following each other, the outer one110 having 1.5 mm diameter on a length of 3 mm, the inner one 111 having800 μm diameter on a length of 1.5 mm. 5 cm silicone capillary tubes 109(Cole-Parmer; outer diameter 1.8 mm, inner diameter 800 μm) coated withsilicon rubber (Dow Corning) are then introduced on 3 mm (correspondingto the outer cylinder) in each hole of the T-connector, and theconnector is strengthened by adding more rubber around the tube near theentry of the PDMS T. Due to silicon and PDMS elasticity, it is possibleto push the 1.8 mm outer diameter silicon tubing in the 1.5 mm diameterhole of the T, thus providing a good tightness of the junction. Theobtained connector is put in the oven for two hours.

The T-connector 100 with silicon capillary tube is further silanisedwith the method described in Example 14. A solid connector with very lowdead volume and no leakage even under high pressure is obtained withthis method. Furthermore, the use of silicon tube allows using pinchvalves which have no dead volume.

EXAMPLES OF APPLICATIONS

A device made in accordance with the invention may be used to carry out,for instance:

-   -   mixing,    -   nucleic acid screening,    -   nucleic acid amplification, e.g. by PCR, NASBA, rolling, circle        amplification    -   RNA reverse transcription    -   genotyping,    -   proteomic analysis,    -   transcriptome analysis,    -   crstallization, and in particular protein crystallisation,    -   searching and evaluation of pharmaceutical targets,        pharmaceutical bits or leads, or drugs,    -   enzyme-protein reaction,    -   antigen-antibody reaction,    -   screening of libraries of chemical of biological products,    -   high throughput screening,    -   drug delivery,    -   diagnosis,    -   analysis or lysis of at least one living cell or dead cell,    -   analysis of microorganisms,    -   chemical reaction,    -   reactive-catalyzer reaction,    -   polymerization reaction,    -   fusing particles, for example colloids, to form a chain,    -   preparation of colloids, emulsions, vesicles, in particular        monodisperse colloidal objects,    -   preparation of nanoparticles or microparticies,    -   environmental control,    -   detection of pollutants,    -   control of an industrial process.

1. A device for performing a PC comprising: a microchannel comprising acoil made of a capillary tube at least partly filled of a packetcomprising a surfactant, the coil comprising a denaturing region, anannealing region and an elongation, region exposed to differenttemperatures.
 2. The device according to claim 1, comprising a cylindercomprising three regions corresponding to the denaturing, annealing andelongation regions.
 3. The device according to claim 2, the capillarytube being wound around the cylinder.
 4. The device according to claim2, the denaturing, annealing and elongation regions being isolated onefrom another by sheets, which are affixed between the pieces of thecylinder.
 5. The device according to claim 1, the device having acylinder shape and comprising three ventilation holes per quartercylinder, said holes being drilled through the entire device to providevents for air cooling.
 6. The device according to claim 5, furthercomprising a turbine configured to blow ambient air through theventilation holes providing better temperature control and uniformity.7. The device according to claim 1, the device having a cylinder shape,two holes fix receiving thermocouples and one central hole for receivinga heater being drilled partially through the cylinder in eachtemperature region, the central hole being larger than the holes forreceiving the thermocouples.
 8. The device according to claim 1, thesize of the elongation region being twice the size of the denaturing andannealing regions.
 9. The device according to claim 1, each regioncomprising a resistance heater and two thermocouples.
 10. The deviceaccording to claim 9, the resistance heaters being located in the centerof their respective region, and the thermocouples being located near theinterface between regions.
 11. The device according to claim 1, thecapillary tube comprising, aqueous droplets.
 12. The device according toclaim 1, the capillary tube comprising a DNA fragment.
 13. The deviceaccording to claim the packet being made of a fluorosolvent.
 14. Amethod of PCR amplification using the device according to claim 1.